Mp

Immunobiol., vol. 171, pp. 45-56 (1986)

Department of Cell Biology and Genetics, Erasmus University, Rotterdam, The Netherlands

Decreased In Vivo Functional T Cell Capacity in the Murine Autoimmune Strains MRL/Mp-Ipr/lpr and Male BXSB/Mp TH.

W. VANDEN AKKER, M. C. 5TUY, A. T.]. BIANCHI, and R. BENNER

Received May 14, 1985 . Accepted October 7, 1985

Abstract We have studied the functional and proliferative capacity of the T cells of systemic lupus erythematosus (SLE)-prone MRL and BXSB mice during aging. The study was performed in vivo, in the delayed-type hypersensitivity (DTH) assay, and in vitro, in the mixed lymphocyte reaction (MLR). Both assays showed depressed T cell responses in MRLll and male BXSB mice at 4 to 5 and 9 to 10 months of age, respectively, when a significant proportion of the animals showed clear-cut disease. At younger ages, the proliferation-dependent DTH was not affected in MRLll and male BXSB mice. This is in agreement with the reported primary B cell defect of male BXSB mice, but not with the reported early T cell abnormalities in MRLll mice. A subnormal reactivity of the highly proliferating T helper cells or the existence of a T cell subset with normal DTH reactivity might account for the relatively long-lasting normal DTH reactivity in MRLll mice.

Introduction The two murine strains MRL and BX5B developed by MURPHY and ROTHS (1) spontaneously develop an autoimmune disease similar to human systemic lupus erythematosus (5LE). The 5LE syndrome occurring in these mice is characterized by a generalized B cell hyperreactivity which is manifested by autoantibody production, high levels of serum immunoglobulins (Ig), increased numbers of Ig-secreting cells and resistance to induction of tolerance (2, 3, 4). The MRL strain can be divided in two substrains, MRLlMp]-lpr/lpr (MRLlI) and the con genic partner MRLlMp]-++I++ (MRLln). MRLlI carries the autosomal recessive gene (lpr) that results in massive T cell proliferation and acceleration of autoimmune disease. The MRLlI strain has early disease in both sexes with a 50 % mortality that has been described to occur at about 5.5 months of age (5). MRLln does not carry the lpr gene and develops a late-life variant of the same disease in both sexes with a 50 % mortality at 18-20 months of age. Abbreviations: BSA = bovine serum albumin; BSS = balanced salt solution; cpm = counts per minute; DTH = delayed type hypersensitivity; 3H-TdR = tritiated thymidine; IL 2 = interleukin-2; MLR = mixed lymphocyte reaction; NMS = normal mouse serum; PBS = phosphate-buffered saline; S.c. = subcutaneously; SLE = systemic lupus erythematosus

46 . TH. W. VAN DEN AKKER, M. C. STUY, A. T.

J.

BIANCHI, and R. BENNER

In the BXSB strain, the onset and severity of the disease are determined by sex. The male mice develop early disease with a reported 50 % mortality at 6 months; the female mice develop late-life disease with a reported 50 % mortality at 20 months of age (5). The etiology and pathogenesis of SLE in mice and humans are still unknown. The disease seems to be multifactorial and is influenced by genetic, virologic, hormonal and immunologic factors (2, 6). Many aspects of the immune system have been studied in order to elucidate the pathogenesis of the autoimmune disease in the MRL and BXSB strains. Primary genetic or acquired abnormalities of B cells and malfunction of the T cell immunoregulatory circuit have been suggested as playing a key role in the pathogenesis of autoimmunity (2, 7, 8). Although MRL and BXSB mice show similar disease patterns, the cellular basis of the condition may be different (6). BXSB male mice display pronounced B cell hyperreactivity, which has been traced to a defect in the hemopoietic stem cells that give rise to B lymphocytes. This defect is assumed to be the major abnormality in the BXSB male mice (9, 10). Recently it was reported that neonatal thymectomy accelerates this B cell hyperreactivity (11), but this finding has not been confirmed (12). In the MRLlI mice, a more direct role of the T cells in the disease has been implicated. The MRLlI mice have a large number of lymphocytes in their lymph nodes that express low levels of the T cell antigens Thy-l and Lyt-l (13). Neonatal thymectomy prevents the generation of these T cells to a large extent and significantly delays the onset and reduces the severity of the autoimmune disease (14, 15). Here we have investigated the functional capacity of the T cells of MRL and BXSB mice during aging in order to gain insight into the possible significance of deterioration of the T cell system in the autoimmune disease in these strains. This was done in vivo, in the delayed-type hypersensitivity (DTH) assay, and in vitro, in the mixed lymphocyte reaction (MLR). We report a decrease in the in vivo functional capacity of the T lymphocytes of MRLlI and male BXSB mice at 4 to 5 and 9 to 10 months of age, respectively, which is most likely due to a defective proliferative capacity.

Materials and Methods Mice Male and female BXSB/MpJ (H-l b) and female MRLlMpJ-Jpr/Jpr(MRLlI) and MRLlMpJ++1++ (MRLln) mice (both H-l k), 4-8 week old, were purchased (November 1981 till January 1983) from The Jackson Laboratory, Bar Harbor, Maine, U.S.A. Male and female C57BLl10.ScSn (H-l b) and female C3H/HeJ (H-2k) mice, 8-12 week old, were obtained from the Laboratory Animals Center of the Erasmus University, Rotterdam, The Netherlands.

Decreased T Cell Capacity in MRL and BXSB . 47 Female DBA12 (H-2 d) mice, 10-12 week old, were purchased from the Radiobiological Institute TNO, Rijswijk, The Netherlands. All mice were kept in our own animal facilities and were allowed free access to food and acidified water.

Preparation of cell suspensions The mice were killed by exposition to carbon dioxide vapor. Immediately after killing, the spleens were removed, placed in a balanced salt solution (BSS) and squeezed through a nylon gauze filter to provide a single cell suspension. Nucleated cells were counted with a Coulter counter model BZI (Coulter Electronics Ltd., Harpenden, Herts, England). Viable nucleated cells were counted in a hemocytometer using 0.1 % fluorescein diacetate in phosphatebuffered saline (PBS) as a diluent.

Antigen and immunization Primary immunization was performed with 1 x 107 allogeneic spleen cells in a volume of 0.1 ml of BSS. The cells were injected subcutaneously (s.c.) and were equally distributed over both inguinal areas by means of a 25-gauge needle.

Assay for DTH

DTH reactions were determined by measuring the difference in thickness of the hind feet 24 h after s.c. injection of 2 x 10 7 allogeneic spleen cells into the dorsum of the right hind foot. The challenge dose was administered s.c. in a volume of 0.05 ml by means of a 25-gauge needle. A control group, consisting of non-immune mice challenged with the same number of spleen cells as the mice to be tested, was always included. The thickness of the left and right hind feet was measured with a footpad meter with a 0.05 mm accuracy. During the measurement, the mice were anesthetized with ether. The specific increase in foot thickness was calculated as the relative increase in foot thickness of the immune mice minus the relative increase in foot thickness of the control mice. The swelling of the control mice ranged between 19 and 29 %. Mixed lymphocyte reactions (MLR) MLR was performed in 96-well culture plates (Greiner, Nurtingen, W. Germany) in a volume of 0.2 ml RPMI 1640 supplemented with 2 % (v/v) normal mouse serum (NMS), 50 units/ml penicillin, 50 !-Ig/ml streptomycin,S x 10- 4 % (v/v) 2-mercaptoethanol and 1 X 10- 3 M sodium pyruvate. Responder and stimulator cell populations were cultured in equal numbers, 1 X 106 cells of each. Stimulator cells were irradiated with 15 Gy X-irradiation generated in a Philips-Muller MG 300 X-ray machine. Cultures were incubated at 37°C in humidified incubators supplied with a mixture of 95 % air and 5 % CO 2• To assay DNA synthesis in MLR, 1.0 !-ICi of tritiated thymidine CH-TdR; specific activity 5.0 Ci/mmol; The Radiochemical Centre, Amersham, England) was added to the cultures for the final 20-22 h of a 4 day incubation period or at relevant times in kinetic experiments. Cultures were harvested onto glass fiber filter paper (Cryoson, Midden-Beemster, The Netherlands) with a semiautomatic multiple sample harvester. Dry filters were placed in a toluene-based scintillation fluid containing 2,5-diphenyloxazole (PPO) and 1,4-bis-2(5-phenyloxazolyl)-benzene (PO POP), and radioactivity was measured in a liquid scintillation counter (Tri-carb 300C, Packard Instrument Corp., IL, U.S.A.). Data are expressed as mean counts per minute (cpm) of nine replicate cultures ± 1 SEM.

Immunofluorescence staining of T cell subsets Monospecific antibodies against Thy-I, Lyt-l and Lyt-2 determinants were obtained from the tissue culture supernatant of the appropriate hybridomas. Anti-Thy-l was derived from clone 59AD2.2, anti-Lyt-1 from clone 53-7.3.13 and anti-Lyt-2 from clone 53-6.72. These antibodies were kindly provided by Dr. W. van Ewijk from our department. All clones were originally produced and characterized by Dr. J. A. Ledbetter, Genetic Systems Inc., Seattle, WA, U.S.A. (16).

48 . TH. W. VAN DEN AKKER, M. C. STUY, A. T.

J.

BIANCHI, and R. BENNER

Spleen cell suspensions were kept in PBS supplemented with 1 % bovine serum albumin (BSA-PBS; pH 7.8). Samples of 1 X 106 cells, in aliquots of 100 Ill, were incubated for 30 min at 4°C with 20 III of the appropriate antibodies. Subsequently, the cells were centrifuged and repeatedly washed in 1 % BSA-PBS. As a second step, 100 III of fluorescein-conjugated rabbit anti-rat serum (RaRalg-FITC; Nordic, Tilburg, The Netherlands) were applied, and the cells were incubated for another 30 min at 4°C. The cells were washed twice with 1 % BSA-PBS, suspended in PBS-glycerol, were mounted on a microscope slide and examined with a fluorescence microscope. Data are expressed as the mean percentage of positive cells ± 1 SEM calculated from at least 3 individual experiments.

Results We have studied the functional capacity of the T cells of MRL and BXSB mice in vivo, in the DTH assay, and in vitro, in the MLR. This was done at three different ages, at 1.5 to 2 months «
80%

50%

0%

~---r----'-~-r----,----.--~r====r====~---+--~.----.-

o

3

6

9

12

15

18

21

AGE (in months)

Fig. 1. Mortality curves of the MRL and BXSB mice which were used.

27

30

33

Decreased T Cell Capacity in MRL and BXSB . 49

cut disease of the group under investigation. This is supported by our finding that at that time point, 80 % of the mice displayed at least 3 mg protein per ml urine (data not shown).

Functional capacity of T lymphocytes in vivo as determined in the DTH assay Primary DTH responses of the MRL and BXSB mice as well as those of their non-autoimmune age-, sex- and H-2-matched controls, C3H and C57BLl10 mice, respectively, were studied at the three ages mentioned above. The mice were s.c. immunized with H-2 and non-H-2 incompatible DBA12 spleen cells and were challenged with similar DBA/2 spleen cells 6 days later. DTH responses were determined 24 h after challenge. Figure 2, upper part, shows that in the MRLlI mice DTH responses could be induced in the «young» age group, although the responses were lower than those of «young» MRLln mice. The «adult» age MRLlI group did not show any DTH response. «Old» MRLlI mice could not be tested in the DTH assay because the size of this group at the age of 9-10 months was too small for experiment. The MRLln and the control mice showed DTH reactivity at all ages tested, but peak responses were measured in the young age group.

4 - 5 months

1. 5 - 2 months

9 - 10 months

MRLlI MRLln C3H

BXSB BXSB

" ~

C57BL " C57BL ~

o

20

40

0

20

40

0

20

40

%SPECIFIC INCREASE FOOT THICKNESS

Fig. 2. Primary DTH responsiveness of female MRLll and MRLln mice (upper part) and male and female BXSB mice (lower part) at 1.5 to 2, 4 to 5 and 9 to 10 months of age. C3H and C57BLllO mice were used as controls. DTH reactivity was induced by s.c. immunization with 1 X 10 7 H-2 and non-H-2 incompatible DBAI2 spleen cells. Six days after immunization, all mice were challenged. 24 h later, the foot swelling was measured. Each column represents the arithmetic mean of the DTH response ± 1 SEM of at least two different experiments (n > 24).

50 . TH. W. VAN DEN AKKER, M. C. STUY, A. T.

J.

BIANCHI, and R. BENNER

The BXSB male mice showed DTH reactivity at the «young» and «adult» age, but decreased DTH responses in the «old» mice. The BXSB female mice and the control mice showed DTH reactivity at all ages tested (Fig. 2, lower part), but gradually decreasing in height with increasing age.

Proliferative capacity of T lymphocytes in vitro as determined in the MLR assay The proliferative responses of 1 X 106 responder cells from MRL and BXSB mice against 1 X 106 allogeneic DBA/2 spleen cells were studied in vitro in the MLR assay. First of all, the kinetics of the proliferative reponse were determined. Cells from MRLlI, MRLln, BXSB male and female and the non-autoimmune control mice showed the same kinetics with peak responses on day 4 at all ages tested (data not shown). Therefore, in all subsequent MLR experiments, the proliferative activity was determined on day 4. The proliferative responses of the BXSB and MRL mice in the MLR were studied at the same age as the mice used for the DTH assay. «Young» MRLlI cells responded in the MLR as well as the control C3H mice. The responsiveness of MRLlI cells derived from «adult» animals was significantly lower. MRLln mice exhibited strong MLR at all ages tested (Fig.3, upper part). The BXSB male mice showed normal proliferative

MRLlI MRLln

f-l

C3H

BXSB BXSB C57BL C57BL

I

d

m

ITl

~

:---

~ ,"I ~

~~

~

J

~

I

o

20

40

I

r---,

1----1

~

~

J-t

N

P-~

~

~

d

9 - 10 months

4 - 5 months

1. 5 - 2 months

10.......--'

60

o

20

40

60

CPM x 10

o

20

40

60

3

Fig. 3. Mixed lymphocyte reaction by spleen cells from female MRLlI and MRLln mice (upper part) and male and female BXSB mice (lower part) at 1.5 to 2, 4 to 5 and 9 to 10 months of age. Age-, sex- and H-2-matched C3H and C57BLl10 were used as controls. Each column represents the arithmetic mean of the response in cpm ± 1 SEM of at least two individual experiments (n > 18). The hatched area represents the mean background 3H-TdR incorporation in cpm ± 1 SEM of control cultures with irradiated syngeneic «stimulator» cells.

Decreased T Cell Capacity in MRL and BXSB . 51

responses at the «young» and «adult» age but a relatively low MLR at the «old» age. BXSB female mice showed a similar responsiveness as the control C57BL mice at all ages tested (Fig. 3, lower part).

T cell subsets in the spleen of MRL and BXSB mice «Adult» MRLlI and «old» BXSB male mice had marked splenomegaly and therefore might have had an altered cellular composition of the spleen. We investigated whether the decrease in responsiveness in the MLR and DTH assay might be related to a decrease in the proportion of responder cells bearing the Lyt-l surface-antigen. Therefore, in the spleens of all groups of mice, the percentages of cells were determined bearing the Thy-I, Lyt-l and Lyt-2 surface marker. Figure 4 (upper part) shows that no % positive cells 50

BXSB

40 30

"y ~ "a ~ " ~ 0

Thy-l

% positive cells 50

"y ~ "a ~ " ~

"y ~ "a ~ " ~

Lyt-l

Lyt-2

0

MRL

0

~

40 30

20 10

I n Inn y a 0 Thy-l

I n Inn y a 0

I n Inn y a 0

Lyt-l

Lyt-2

Fig. 4. Percentages of Thy-1 +, Lyt-1 + and Lyt-2+ cells in the spleens of female MRLll and MRLln mice and male and female BXSB mice, as determined by immuno-fluorescence with monoclonal antibodies. Below each diagram the age of the mice used is indicated as follows: «young» (y), «adult» (a) and «old» (0). Each column represents the mean percentage of positive cells ± 1 SEM, calculated from 3 individual experiments.

52 . TH.

W. VAN DEN AKKER, M. C. STUY, A. T.

J.

BIANCHI,

and R. BENNER

significant differences were found in the percentages of Lyt-1 + and Lyt-2+ cells in the BXSB male and female mice at all ages tested. The percentages of Thy-1 + cells from the «young» and «adult» BXSB female mice were somewhat higher than those of age-matched BXSB male mice, but the difference was no longer significant in the «old» mice. The MRLlI mice were found to have higher percentages of Thy-1 + cells than MRLln mice, which in contrast to MRLln, increased with age (Fig. 4, lower part). MRLlI and MRLln displayed comparable percentages of Lyt1+ cells, although the percentage of Lyt-1 + cells tended to increase with age in the MRLlI mice. The percentage of Lyt-2+ cells in MRLlI mice decreased with age. In MRLln mice, the frequency of Lyt-2+ cells remained constant. The percentages Thy-1 +, Lyt-1 + and Lyt-2+ cells of the MRLlI and MRLln were higher than those of the BXSB male and female mice, which correlated with the significantly higher 3H -T dR incorporation in MLR by spleen cells from MRL mice (Fig. 3).

Discussion This paper represents an integrated study of the mortality rate, T cell subset distribution and functional activity of T cells of MRL and BXSB mice at various ages. The latter were determined at 1.5-2 «
Decreased T Cell Capacity in MRL and BXSB . 53

autoimmune disease, have the best chances for procreation, thus selecting long-living mice and excluding the autoimmune trait in time. An alternative explanation is that the better survival of BXSB males is due to our (cleanconventional) housing conditions. However, MRLlI mice which were kept under the same housing conditions did not show a similar effect with regards to their survival. Most proliferating T cells in the MLR bear the surface marker Lyt-1 or L3T4 (18, 19). DTH reactivity, which is also predominantly mediated by Lyt-1 + cells (20, 21), is dependent on proliferation of the DTH reactive cells (22, 23) and on the production of lymphokines, which are responsible for the inflammatory lesion (24), as well as on mononuclear and polymorphonuclear phagocytic cells (24). The coincidence of depressed responses in the MLR and DTH assay in «adult» MRLlI and «old» male BXSB mice, together without decreasing frequency of Lyt-1 + cells, suggests that the decreased DTH responses are due to a defective proliferation of antigenspecific Lyt-1 + cells. The relative unresponsiveness in the MLR of the «adult» MRLlI mice, which was also found by others (25), and «old" male BXSB mice was not due to a lack of Lyt-1 + cells: The «adult» MRLlI and «old» BXSB male mice had a marked splenomegaly, but the percentages of Lyt-1 + cells were comparable to «adult» MRLln and «old» female BXSB mice, respectively. Our results concerning the quantitation of T cell subsets in the spleen (Fig.4) grossly agree with those reported by others (25). MORSE et al. (26) reported somewhat higher percentages of Lyt-1 + cells in the spleen. However, they used a fluorescence-activated cell sorter and, thus, counted both the brighter and the duller Lyt-1 + cells. Using immunofluorescence microscopes, we could not reliably count most of the dull Lyt-1 + cells. The decreasing percentages of Lyt-2+ cells in aging MRLlI mice agree with MORSE et al. (26). They reported that the effect of the lpr mutation on spleen cells is less marked than on lymph node cells. Our experiments cannot conclusively answer the question of whether the decreased DTH response is solely due to a defective proliferation of the DTH effector T cells or whether there is also a defective production of lymphokines by the DTH effector T cells and/or a deficiency in the mononuclear and polymorphonuclear phagocytic cell populations. In contrast to previous reports, it was recently shown that T cell lines derived from MRLlI spleens could produce interleukin-2 (IL 2) and respond to IL 2 (27). The fundamental lesion that leads to the development of autoimmunity in MRLlI mice could be the inability to regulate the activity of interferon: high production of interferon causes enhanced Ia expression on resting macrophages, which might initiate the autoimmune response (28). Defects in MRLlI macrophage activity, as measured by Ia expression and by levels of interleukin-1 production, could not be detected by Lu and UNANUE (29). SCOTT et al. (30) found that defects in proliferative cell responses in MRL/I mice were not due to inadequate antigen presentation

54 . TH. W. VAN DEN AKKER, M. C. STUY, A. T.

J. BIANCHI, and R. BENNER

by macrophages. THEOFILOPOULOS et al. (31), however, reported MRLlI spleen cells to be poor stimulator cells as compared to MRLln mice in a cytotoxic T cell response. Our finding that adult MRLlI mice, in contrast to MRLln, are unable to generate allogeneic DTH reactions is in agreement with that of others (30), who could not find TNP-specific DTH in MRLlI, in contrast to MRLln. However, they did find a significant footpad swelling at 4-h post-challenge in their MRLlI mice, indicating an Arthus-type hypersensitivity. Here it must be considered that the depressed responses in the DTH and MLR assay in «adult» MRLlI and «old» BXSB male mice may represent a generalized debilitation of immunological functions in moribund SLE mice. In view of data showing that predominantly T helper cells account for DTH (32, 33), it is remarkable that our study shows DTH hypo reactivity accompanied by MLR hyporeactivity, despite normal to increased numbers of Lyt-1 positive cells. Thus, either these T helper cells have subnormal DTH reactivity, or alternatively, a T cell subset exists with normal DTH reactivity, which is gradually ousted by the expanding pool of proliferating Lyt-1 positive T cells that by themselves are incapable of DTH reactivity. Further studies are necessary to differentiate between these alternatives. Acknowledgement We gratefully acknowledge Mrs. CARY MEIJERINK-CLERKX for typing the manuscript.

References

J. B. ROTHS. 1978. Autoimmunity and Iymphoproliferation: induction by mutant gene Ipr, and acceleration by a male-associated factor in strain BXSB mice. In: Genetic Control of Autoimmune Disease. ROSE, N. R., P. E. BIGAZZI and N. L. WARNER, (eds.). Elsevier/North-Holland, New York, p. 207. THEOFILOPOULOS, A. N., and F. J. DIXON. 1981. Etiopathogenesis of murine SLE. Immunol. Rev. 55: 179. ANDREWS, B. S., R. A. EISENBERG, A. N. THEOFILOPOULOS, S. IZUI, C. B. WILSON, P. J. MCCONAHEY, E. D. MURPHY, J. B. ROTHS, and F. J. DIXON. 1978. Spontaneous murine lupus-like syndromes. J. Exp. Med. 148: 1198. LASKIN, C. A., J. D. TAUROG, P. A. SMATHERS, and A. D. STEINBERG. 1981. Studies of defective tolerance in murine lupus. J. Immunol. 127: 1743. DIXON, F. J. 1981. Murine systemic lupus erythematosus. Immunol. Today 2: 145. STEINBERG, A. D., D. P. HUSTON, J. D. TAUROG, J. S. COWDERY, and E. S. RAVECHE. 1981. The cellular and genetic basis of murine lupus. Immunol. Rev. 55: 121. KNIGHT, J. G. 1982. Autoimmune diseases: defects in immune specificity rather than a loss of suppressor cells. Immunol. Today 3: 326. CANTOR, H., and R. K. GERSHON. 1979. Immunological circuits: cellular composition. Fed. Proc. 38: 2058. EISENBERG, R. A., S. IZUI, P. J. MCCONAHEY, L. HANG, C. J. PETERS, A. N. THEOFILOPOULOS, and F. J. DIXON. 1980. Male determined accelerated autoimmune disease in BXSB mice: transfer by bone marrow and spleen cells. J. Immunol. 125: 1032. HANG, L., S. IZUI, J. H. SLACK, and F. J. DIXON. 1982. The cellular basis for resistance to induction of tolerance in BXSB SLE male mice. J. Immunol. 129: 787.

1. MURPHY, E. D., and

2.

3.

4. 5.

6.

7. 8.

9. 10.

Decreased T Cell Capacity in MRL and BXSB . 55 11. SMITH, H. R., T. M. CHUSED, P. A. SMATHERS, and A. D. STEINBERG. 1983. Evidence for thymic regulation of autoimmunity in BXSB mice: acceleration of disease by neonatal thymectomy. J. Immunol. 130: 1200. 12. HANG, L., A. N. THEOFILOPOULOS, R. S. BALDERAS, S. J. FRANCIS, and F. J. DIXON. 1984. The effect of thymectomy on lupus-prone mice. J. Immunol. 132: 1809. 13. LEWIS, D. E., J. V. GIORGI, and N. L. WARNER. 1981. Flow cytometry analysis of T cells and continuous T cell lines from autoimmune MRLlI mice. Nature 289: 298. 14. WOFSY, D., J. A. LEDBETTER, J. R. ROUBINIAN, W. E. SEAMAN, and N. TALAL. 1982. Thymic influences on autoimmunity in MRL-Ipr mice. Scand. J. Immunol. 16: 51. 15. STEINBERG, A. D., J. B. ROTHS, E. D. MURPHY, R. T. STEINBERG, and E. S. RAVECHE. 1980. Effects of thymectomy or androgen administration upon the autoimmune disease of MRLlMp- lprllpr mice. J. Immunol. 125: 871. 16. LEDBETTER, J. A., and L. A. HERZENBERG. 1979. Xenogeneic monoclonal antibodies to mouse lymphoid differentiation antigens. Immunol. Rev. 47: 63. 17. MOSCOVITCH, M., E. ROSENMANN, Z. NEEMAN, and S. SLAVIN. 1983. Successful treatment of autoimmune manifestations in MRLlI and MRLln mice using total lymphoid irradiation (TLI). Exp. Mol. Path. 38: 33. 18. CANTOR, H., and E. A. BOYSE. 1975. Functional subclasses of T lymphocytes bearing different Ly antigens. I. The generation of functionally distinct T-cell subclasses is a differentiative process independent of antigen. J. Exp. Med. 141: 1376. 19. DIALYNAS, D. P., D. B. WILDE, P. MARRACK, A. PIERRES, K. A. WALL, W. HAVRAN, G. OTTEN, M. R. LOKEN, M. PIERRES,J. KAPPLER, and F. W. FITCH. 1983. Characterization of the murine antigenic determinant, designated L3T4a, recognized by monoclonal antibody GK-1.5: expression of L3T4a by functional T cell clones appears to correlate primarily with class II MHC antigen-reactivity. Immunol. Rev. 74: 29. 20. VADAS, M. A., J. F. A. P. MILLER, I. F. C. McKENZIE, S. E. CHISM, F. W. SHEN, E. A. BOYSE, J. R. GAMBLE, and A. M. WHITELAW. 1976. Ly and la antigen phenotypes of T cells involved in delayed-type hypersensitivity and in suppression. J. Exp. Med. 144: 10. 21. BRIL, H., B. D. MOLENDIjK-LOK, L. M. HUSSAARTS-ODIjK, and R. BENNER. 1984. Synergism of T lymphocyte subsets in the response to MIs-locus coded antigens during graft-vs-host reaction. Cell. Immunol. 83: 370. 22. BLOOM, B. R., L. D. HAMILTON, and M. W. CHASE. 1964. Effects of mitomycin C on the cellular transfer of delayed type hypersensitivity in the guinea pig. Nature 201: 689. 23. WOLTERS, E. A. J., and R. BENNER. 1979. Immunobiology of the Graft-versus-Host reaction. II. The role of proliferation in the development of specific anti-host immune responsiveness. Transplantation 27: 39. 24. SCHREIER, M. H., R. TEES, A. A. NORDIN, R. BENNER, A. T. J. BIANCHI, and M. J. VAN ZWIETEN. 1982. Functional aspects of helper T cell clones. Immunobiol. 161: 107. 25. ALTMAN, A., A. N. THEOFlLOPOULOS, R. WEINER, D. H. KATZ, and F. J. DIXON. 1981. Analysis of T cell functions in autoimmune murine strains. Defects in production of and responsiveness to interleukin 2. J. Exp. Med. 154: 791. 26. MORSE III, H. c., W. F. DAVIDSON, R. A. YETTER, E. D. MURPHY,J. B. ROTHS, and R. L. COFFMAN. 1982. Abnormalities induced by the mutant gene lpr: expansion of a unique lymphocyte subset. J. Immunol. 129:2612. 27. ROSENBERG, Y. J., A. D. STEINBERG, and T. J. SANTORO. 1984. T cells from autoimmune «IL-2 defective» MRL-Ipr/lpr mice continue to grow in vitro and produce IL-2 constitutively. J. Immunol. 133: 2545. 28. SANTORO, T. J. 1984. Systemic lupus erythematosus: insights from animal models. In: Cytokines. STEINBERG, A. D., moderator. Ann. Intern. Med. 100: 714. 29. Lu, C. Y., and E. R. UNANUE. 1982. Spontaneous T celllymphokine production and enhanced macrophage Ia expression and tumoricidal activity in MRLllpr mice. Clin. Immunol. Immunopathol. 25: 213. 30. SCOTT, C. F., M. TSURUFUjI, C. Y. Lu, R. FINBERG, and M. S. SY. 1984. Comparison of antigen specific T cell responses in autoimmune MRLlMp-lprilpr and MRLlMp- +1+ mice. J. Immunol. 132: 633.

56 . TH. W. VAN DEN AKKER, M. C. STUY, A. T.

J.

BIANCHI, and R. BENNER

31. THEOFILOPOULOS, A. N., D. L. SHAWLER, D. H. KATZ, and F.]. DIXON. 1979. Patterns of immune reactivity in autoimmune murine strains. 1. Cell-mediated immune responses induced by H-2 identical and H-2 incompatible stimulator cells. J. Immunol. 122: 2319. 32. BIANCHI, A. T. J., H. HOOlJKAAS, R. BENNER, R. TEES, A. A. NORDIN, and M. H. SCHREIER. 1981. Clones of helper T cells mediate antigen-specific, H-2 restricted delayed type hypersensitivity. Nature 290: 62. 33. MILLER, J. F. A. P. 1983. Genetic control of lymphocyte interactions. In: Progress in Immunology. V. YAMAMURA, Y. and T. TADA, eds. Academic Press, Tokyo, p. 797. Dr. Th. W. VAN DEN AKKER, Department of Cell Biology and Genetics, Erasmus University, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands